Despite recent advances in targeted cancer therapeutics, molecular determinants of response to these agents are often unclear and acquired resistance is widespread. Effective treatment of cancer patients necessitates an understanding of these mechanisms. Highly specific cyclin-dependent kinase 4 and 6 (CDK4 and CDK6) inhibitors represent a novel and exciting class of targeted agents. CDK4/6 inhibitors prevent cell cycle progression from the first growth phase (G1) to the DNA synthesis (S) phase of the cell cycle. Although CDK4/6 inhibitors are the most advanced clinically in breast cancers that express the estrogen receptor (ER+), the mechanisms determining response and resistance are not fully characterized. Prior studies suggest that ER+ breast cancer cells that are resistant to estrogen deprivation therapy (a mainstay of treatment for this subtype) exhibit a dependency on CDK4 in vitro and in vivo. Furthermore, we have found that breast cancer cells expressing a constitutively active (and estrogen-independent) ER mutant are exquisitely sensitive to CDK4/6 inhibitor treatment. Therefore, we hypothesize that use of CDK4/6 inhibitors in breast cancer cells that are dependent on ER signaling might enrich for sensitivity to CDK4/6 inhibition. Most in vitro resistance studies on CDK4/6 inhibitors have only focused on cell cycle genes. It is unknown whether other pathways can also mediate resistance to CDK4/6 inhibition. The ultimate goal of the proposed research is to identify clinical predictors of response to CDK4/6 inhibitors and to preserve the efficacy of these targeted agents. To accomplish this goal, two proposed aims seek to (1) determine whether ER-mutant breast cancer cells are sensitive to CDK4/6 inhibition (2) systematically identify genes that can confer resistance to CDK4/6 inhibition in ER+ breast cancer.